Mass spectrometry has been used for many decades in the characterization of small organic molecules. The technique typically involves the ionization of molecules in the sample to form molecular ions by subjecting the sample to an electron beam at a very low pressure (10−5 to 10−6 torr). The molecular ions are then focused and accelerated by an electric field into a magnetic field or quadrapole. The ions are separated in the magnetic field or quadrapole according to the ratio of the mass of the ion m to the charge on the ion z (m/z). After passing through the field, the ions impinge upon a detector which determines the intensity of the ion beam and the m/z ratio, and these data are used to create the mass spectrum of the sample.
With the increasing interest in larger molecules, especially biomolecules such as nucleic acids and proteins, new techniques in the field of mass spectrometry are continually being developed to characterize these molecules. Currently, mass spectrometry of biomolecules is typically done by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) or electrospray mass spectrometry. However, most large biomolecules such as oligonucleotides are poly-ionic. In electrospray mass spectrometry, the poly-ionic nature of these molecules results in multiply charged ions which complicate the interpretation of the mass spectrum. In MALDI-TOF mass spectrometry, the ions are mostly singly charged; therefore, during the ionization process all except for one charge on the molecule must be neutralized. The efficiency of the ionization process for polyionic molecules is therefore reduced.
A need exists for techniques in mass spectrometry which increase sensitivity and/or selectivity in the analysis of polyionic molecules. One way to accomplish this is through the use of mass ionization tags. These tags contain functionalities that will increase the effectiveness of the ionization process and/or make the process more selective. Mass ionization tags are typically covalently attached to the molecule which is to be ionized and analyzed by mass spectrometry.
Several groups have devised mass ionization tags with positively charged quaternary ammonium groups. Aebersold et al. have used a quaternary ammonium group to enhance the ionization of N-phenylthiohydantoins (PTH) of amino acids, which are formed during the Edman degradation of proteins or polypeptides (Aebersold et al., Protein Science 1:494-503, 1992). The use of the novel Edman-type sequencing reagent 3-[4′(ethylene-N,N,N-trimethyl-amino)phenyl]-2-isothiocyanate results in unusually high ionization efficiencies which suppress chemical noise by selectively enhancing detection of the quaternary amine-containing compounds.
The use of cleavable tags that contain quaternary ammonium groups or other amine groups in order to increase their ionization efficiency has also been described for use in identifying and characterizing biomolecules (Van Ness et al., EP 0850320, 31 Jul. 1997; Van Ness et al., EP 0840804, 31 Jul. 1997; Van Ness et al., EP 0868583, 31 Jul. 1997). Giese et al. have disclosed the use of cleavable electrophore tags (Giese et al., U.S. Pat. No. 5,516,931, May 14, 1996). The incorporation of quaternary ammonium groups in peptides by reaction of amines with quaternary ammonium N-hydroxysuccinimidyl (NHS) alkyl esters and subsequent detection by MALDI-MS has been reported (Bartlett-Jones et al., Rapid Communications in Mass Spectrometry 8:737-742, 1994).
Gut et al. have disclosed the use of backbone alkylation of phosphorothioate-containing oligonucleotides, and more specifically the use of quaternary amine tags in association with backbone alkylation in order to increase sensitivity and selectivity (Gut et al., Rapid Commun. Mass. Spectrom. 11:43-50, 1997; Gut et al., Nucleic Acids Res. 23: 1367-1373, 1995; Gut et al., WO 96/27681, 12 Sep. 1996). In this method, the increased selectivity for the oligonucleotides of interest is presumed to result from (1) these molecules having one pre-made positive charge and no negative charges whereas the other oligonucleotides in the sample are multiply charged, and (2) the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric system being less sensitive for multiply charged ions. For example, MALDI-TOF mass spectroscopy has a higher sensitivity for proteins than for oligonucleotides of similar molecular weight. Disadvantages of this method include incomplete alkylation of the backbone or overalkylation of the oligonucleotide with hydroxyl groups of the sugar or amines of the nucleotide bases being alkylated. Varying degrees of alkylation in the sample lead to difficulty in interpreting the mass spectrum due to the different molecular weights of the various alkylated oligonucleotides.
A method has also been reported to improve ionization and reduce fragmentation of polyionic analytes using polyionic reagents that form non-covalent complexes with the analytes (Biemann et al., U.S. Pat. No. 5,607,859, Mar. 4, 1997). These polyionic reagents are of opposite charge to the analyte resulting in complex formation. Under soft ionization conditions, the complex with the analyte and reagent together can be ionized resulting in stabilization of the ion. However, this method results in mass spectral peaks that contain no adducts, one adduct, or multiple adducts thereby complicating interpretation of the mass spectrum. Furthermore these complexes are formed using strongly basic or acidic reagents that rely on extremes of pH to achieve a charge, rather than incorporating permanently charged groups such as quaternary ammonium functionalities.
These techniques described in the prior art rely on two principles. The first is the tendency of certain functionalities within the tag to readily pick up charge, and the second is the use of tags consisting of functional groups that are “pre-charged” and do not undergo formal ionization (i.e., quaternary ammonium groups). An example of a tendency to readily pick up charge is amines with APCI (atmospheric chemical ionization) mass spectrometry, and an example of “pre-charged” tags is quaternary ammonium functionalities with ion evaporation mass spectrometry or MALDI-MS.
Although these processes can be effective, as in the case of oligonucleotide backbone alkylation, the oligonucleotide must be synthesized starting from special modified nucleotides in which one of the oxygens on the phosphate is replaced by a sulfur atom. The process of alkylation itself requires extra steps and manipulation of the sample. Also, this process of alkylation must be finely controlled in order to minimize the occurrence of over-alkylation, which if it occurs can make interpretation of the mass spectrum more difficult.
In summary, previous use of mass ionization tags attempt to achieve a single charge by 1) reacting the charges with alkylating agents, 2) neutralization of the charges through proton transfer with the matrix, 3) using a single readily ionizable or permanently charged functionality, such as a quaternary ammonium group, or 4) noncovalent complexation of a multiply charged polyionic reagent with the polyionic analyte. These methods frequently require modified analytes and extra sample preparation steps. And in the end, they can result in difficulties in mass spectrum interpretation due to low ionization efficiency, fragmentation of the molecular ion, or the formation of multiple adducts. There remains a need for improved methods to increase the ionization efficiency and sensitivity/selectivity of polyionic molecules in mass spectrometry.